This application builds on our recent innovations involving a novel combination of magnetic resonance (MRS) and optical (OS) spectroscopic approaches for studying mitochondrial energetics in human muscle. Muscle provides a unique window on the mitochondrial defects that are at the center of a growing number of metabolic and degenerative diseases (e.g., diabetes, neurodegeneration and aging). In this competing renewal, we use the dual OS/MRS spectroscopy approach applied to skeletal muscle to develop diagnostic procedures for identifying defects in mitochondrial function. The proposed studies build on progress made over the tenure of this grant that have led to two key achievements: 1) a combined OS/MRS strategy for measuring chemical content and dynamics that yield energy fluxes and 2) new optical analytical tools that open the visible spectrum for exploitation in vivo. This progress has led to the first quantitative measurement of Oz uptake, which combined with ATP flux, measures mitochondrial coupling non-invasively in vivo. Aim #1 expands on this mitochondria! functional measurement and capitalizes on our ability to use the visible spectrum to develop a measurement of mitochondrial capacity based on cytochrome aas content. To achieve this, we will exploit new analytical methods that permit extending our optical spectroscopy into the visible wavelengths. Aim #2 combines new optical and MRS methods to determine cellular and vascular oxygenation in vivo. A novel optical wavelength shift analysis combined with quantification by MRS yields cellular oxygenation ([oxy-Mb]/total [Mb]) and blood oxygenation ([oxy-Hb]/total [Hb]). These oxygenation measurements have the potential to reveal vascular dysfunction, as well as calibrate our O2 uptake determinations. Aim #3 proposes to design, fabricate and implement a multimodal apparatus that will integrate optical spectroscopy with interleaved, multinuclear 1H and 31P MR spectroscopy. This apparatus uses the technological advances in Aims #1 and #2 to permit new measurements and increased signal-to- noise that will allow studies of mitochondrial function in human muscle in a single session of <90 min. Aim #4 translates this diagnostic tool to aging, which is accompanied by significant mitochondrial dysfunction and is a malady of considerable importance to the health of the nation. The ability to conduct such a complete analysis in a clinically feasible session will provide a unique diagnostic probe of the nature and extent of dysfunction in metabolic and degenerative disorders. Upon completion of this project we will be in position to translate our tools to standard wide bore 3T or other human magnets for use in a clinical setting.